Lipid homeostasis is essential to all living beings that rely on lipid membranes to separate their cell's vital functions from the environment, including all animals, and humans. Furthermore, lipids are used as energy reservoirs by many organisms. A vast array of different lipidic substances, including, for example, phospholipids, triglycerides, fatty acids, and sterols, perform a wide variety of essential functions in cells. Altogether, lipid homeostasis is a tightly regulated, multi-branched, intricate web of interdependent processes in essentially all higher organisms.
Naturally, the more complex a system, the more can go awry. A large number of diseases and conditions, e.g. in humans, are known to be, in whole or in part, consequences of lipid homeostasis dysfunctions. These include both inherited diseases, where one or a number of the many genes involved in lipid homeostasis completely or partially loses its function, or is mis-regulated, as well as acquired diseases, where gene function or gene regulation in the body is altered after single or repeated contact with one or a combination of substances.
In many a species including humans, the body's needs for lipids are filled partially by dietary intake as well as by the synthesis of lipids from precursors. The liver stands out as the single organ responsible for the collection of dietary lipid intake, lipid synthesis, and the control of lipid release to and re-uptake from the bloodstream. Consequentially, it is involved in many, if not all, lipid metabolism disorders.
Many such disorders are caused by, or accompanied with, an overabundance of certain lipids in all or parts of the body, be it from excessive intake, faulty degradation or transport, or excessive de novo synthesis.
For example, Non-Alcoholic Fatty Liver Disease (NAFLD) is a condition where excess triglycerides accumulate in the liver, and is associated with various drugs, nutritional factors, multiple genetic defects in energy metabolism, and, most prominently, insulin resistance (Browning J D and Horton J D, J. Clin. Investigation 2004, 114:147). Conversely, a hallmark of atherosclerosis is the appearance of so-called foam cells, macrophages filled with excess cholesterol and cholesterol esters (Knuth H S, Front Biosci 2001, 6:D429). Other non-limiting examples of disorders associated with excessive levels of lipids in the body are: non-alcoholic liver disease, fatty liver, hyperlipemia, hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia and/or hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulin dependent diabetes mellitus (NIDDM), coronary heart disease, obesity, metabolic syndrome, peripheral arterial disease, and cerebrovascular disease. The treatment of disorders of this type could potentially be aided by attenuating the body's own synthesis of lipids.
A central element in the regulation of lipid biosynthesis in the human liver is a group of transcription factors termed Sterol Regulatory Element Binding Proteins (SREBPs). There are three SREBP isoforms called SREBP-1a, SREBP-1c and SREBP-2. They are located in the endoplasmatic reticulum (ER) in a precursor form (Yokoyama C. et al., Cell 1993, 75:187; Hua X. et al., Proc. Natl. Acad. Sci. 1993, 90:11603) which, in the presence of cholesterol, is bound to cholesterol and two other proteins: SCAP (SREBP-cleavage activating protein) and Insig1 (Insulin-induced gene 1). When cholesterol levels fall, Insig-1 dissociates from the SREBP-SCAP complex, allowing the complex to migrate to the Golgi apparatus, where SREBP is cleaved by S1P and S2P (site 1/2 protease; Sakai J et al, Mol. Cell. 1998, 2:505; Rawson R. B. et al, Mol. Cell. 1997, 1:47), two enzymes that are activated by SCAP. The cleaved SREBP then migrates to the nucleus and acts as a transcription factor by binding to the SRE (sterol regulatory element) of a number of genes and stimulating their transcription (Briggs M. R. et al., J. Biol. Chem. 1993, 268:14490). Among the genes transcribed are the LDL-Receptor, up-regulation of which leads to increased in-flux of cholesterol from the bloodstream, HMG-CoA reductase, the rate limiting enzyme in de-novo cholesterol synthesis (Anderson et al, Trends Cell Biol 2003, 13:534), as well as a number of genes involved in fatty acid synthesis.
In an attempt to lower the body's own production of lipids, one attractive option therefore would seem to be the blocking of SREBP activation. Since SCAP-binding is a prerequisite for the transport and activation of all three SREBP isoforms, an inhibition of SCAP's activity could lead to a general down-regulation of cellular lipid synthesis and uptake. For example, SCAP activity could be inhibited by agents binding to the sterol sensing domain (SSD) of SCAP with higher affinity than cholesterol, and preferably in an irreversible manner, thereby prohibiting SREBP transport and activation. Alternatively, inhibiting the translation and/or transcription of the gene encoding SCAP could lead to lower levels of SCAP present in the ER membrane and available for SREBP-binding and activation.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Despite significant advances in the field of RNAi and advances in the treatment of pathological processes mediated by excessive levels of lipids, there remains a need for an agent that can selectively and efficiently attenuate the body's own lipid biosynthesis, e.g by inhibiting SCAP, and thereby SREBP, activity, using the cell's own RNAi machinery. Such agent shall possess both high biological activity and in vivo stability, and shall effectively inhibit expression of a target SCAP gene, such as human SCAP, for use in treating pathological processes mediated directly or indirectly by SCAP expression.